Use of cation-exchange chromatography in the flow-through mode to enrich post-translational modifications

ABSTRACT

The present invention relates to improved methods in the separation recombinant polypeptides with post-translational modifications from complex mixtures through the use of a cation exchange medium.

BACKGROUND OF THE INVENTION

Specific post-translational modifications and impurity levels are oftenmandated by regulatory agencies for recombinant polypeptides intendedfor human administration. Often the level of post-translationalmodifications of recombinant polypeptides produced in cell lines invitro differs from the level of post-translational modificationsrequired for recombinant polypeptides intended for human administration.The glycosylation profile of a polypeptide can be affected by multiplefactors including the type of production cell line, the growthconditions and the polypeptide sequence. Shiestl, M., et al. NatureBiotechnology 29(4):310 (2011).

There is a need in the art for methods that provide for enrichment ofrecombinant polypeptides with specific levels of particularpost-translational modifications on a scale that is amenable formanufacturing processes, i.e. that maintains an acceptable productrecovery and yield.

SUMMARY OF THE INVENTION

The present invention provides a method for enriching the level ofpost-translational modification of recombinant polypeptides, comprising:contacting a composition which comprises an initial population ofrecombinant polypeptides having different levels of post-translationalmodification with a cation exchange chromatography (CEX) medium operatedin a flow-through mode; wherein recombinant polypeptides that do notbind the CEX medium are separated from recombinant polypeptides thatbind the CEX medium, and wherein the recombinant polypeptides that donot bind the CEX medium comprise a higher level of post-translationalmodification compared to the bound recombinant polypeptides.

The present invention provides a method for enriching the level ofpost-translational modification of recombinant polypeptides, comprising:

-   -   a) contacting a composition which comprises an initial        population of recombinant polypeptides having different levels        of post-translational modification with a cation exchange        chromatography (CEX) medium operated in a flow-through mode; and    -   b) separating recombinant polypeptides that do not bind the CEX        medium from recombinant polypeptides that bind the CEX medium;        wherein the recombinant polypeptides that do not bind the CEX        medium that are recovered comprise a higher level of        post-translational modification compared to the bound        recombinant polypeptides.

The present invention provides a method for enriching the level ofpost-translational modification of recombinant polypeptides, comprising:

-   -   a) contacting a composition which comprises an initial        population of recombinant polypeptides having different levels        of post-translational modification with a cation exchange        chromatography (CEX) medium operated in a flow-through mode;    -   b) separating recombinant polypeptides that do not bind the CEX        medium from recombinant polypeptides that bind the CEX medium;        and    -   c) recovering the recombinant polypeptides that do not bind the        CEX medium;        wherein the recombinant polypeptides that do not bind the CEX        medium comprise a higher level of post-translational        modification compared to the bound recombinant polypeptides.

The present invention provides a method for enriching the level ofpost-translational modification of recombinant polypeptides, comprising:

-   -   a) providing a composition which comprises an initial population        of recombinant polypeptides having different levels of        post-translational modification;    -   b) contacting the composition with a cation exchange        chromatography (CEX) medium operated in a flow-through mode;    -   c) separating recombinant polypeptides that do not bind the CEX        medium from recombinant polypeptides that bind the CEX medium;        and    -   d) recovering the recombinant polypeptides that do not bind the        CEX medium;        wherein the recombinant polypeptides that do not bind the CEX        medium comprise a higher level of post-translational        modification compared to the bound recombinant polypeptides.

In some embodiments, the method further provides recovering therecombinant polypeptides that do not bind the CEX medium.

In some embodiments, the post-translational modification is sialylationor gamma-carboxyglutamate (Gla) formation. In some embodiments, thepost-translational modification is sialylation.

In some embodiments, the total sialic acid content of the recombinantpolypeptides that do not bind the CEX medium is between about 0.5 andabout 6 moles of sialic acid per mole of protein higher than that of theinitial population of recombinant polypeptides. In some embodiments, thetotal sialic acid content of the recombinant polypeptides that do notbind the CEX medium is between about 0.5 and about 4 moles of sialicacid per mole of protein higher than that of the initial population ofrecombinant polypeptides. In some embodiments, the total sialic acidcontent of the recombinant polypeptides that do not bind the CEX mediumis between about 5% and about 100% higher than that of the initialpopulation of recombinant polypeptides. In some embodiments, the totalsialic acid content of the recombinant polypeptides that do not bind theCEX medium is between about 5% and about 40% higher than that of theinitial population of recombinant polypeptides. In some embodiments, thetotal sialic acid content of the recombinant polypeptides that do notbind the CEX medium is between about 12 and about 20 moles of sialicacid per mole of protein. In some embodiments, the total sialic acidcontent of the recombinant polypeptides that do not bind the CEX mediumis between about 14 and about 17 moles of sialic acid per mole ofprotein.

In some embodiments, the total sialic acid content of the initialpopulation of recombinant polypeptides is about 10 to about 14 moles ofsialic acid per mole of protein. In some embodiments, the total sialicacid content of the initial population of recombinant polypeptides isabout 13-14 moles of sialic acid per mole of protein.

In some embodiments, the recombinant polypeptide comprises an Fc domain.In some embodiments, the recombinant polypeptide comprises an antibody.

In some embodiments, the recombinant polypeptide comprises an Fc fusionpolypeptide comprising a ligand binding domain of a receptor. In aspecific embodiment, the receptor is a TNF receptor. In a more specificembodiment, the recombinant polypeptide is etanercept.

In some embodiments, the recombinant polypeptide comprises a clottingfactor. In some embodiments, the recombinant polypeptide is amonomer-dimer hybrid. Examples of clotting factors include Factor VII(FVII), FVIIa, Factor VIII (FVIII), Factor IX (FIX), or FIXa (FIX).FVIII can be full-length FVIII or B-domain deleted FVIII. FVIII can besingle chain FVIII or dual chain FVIII.

In some embodiments, the contacting occurs at a load ratio between about30 and about 100 mg total protein/ml CEX medium. In some embodiments,the contacting occurs at a load ratio between about 33 and about 54 mgtotal protein/ml CEX medium. In some embodiments, the contacting occursat a load ratio of at least about 41 mg total protein/ml CEX medium.

In some embodiments, the contacting occurs at a pH between about 4 andabout 7. In some embodiments, the contacting occurs at a pH betweenabout 5 and about 6. In some embodiments, the contacting occurs at a pHbetween about 5.5 and about 5.8 In some embodiments, the contactingoccurs at a pH of at least about 5.6.

In some embodiments, the contacting occurs at a conductivity betweenabout 8 and about 12 mS/cm. In some embodiments, the contacting occursat a conductivity of between about 9.5 and about 11 mS/cm. In someembodiments, the contacting occurs at a conductivity of at least about10 mS/cm.

In some embodiments, the recombinant polypeptides that do not bind theCEX medium comprise about 25% to about 80% of the initial population ofrecombinant polypeptides. In some embodiments, the recombinantpolypeptides that do not bind the CEX medium comprise about 55% to about80% of the initial population of recombinant polypeptides.

In some embodiments, the CEX medium comprises one of the followingligands: sulfoethyl; sulphopropyl; sulfopropyl; CH₂—SO₃ ⁻; CH₂CH₂CH₂SO₃⁻; SO₃ ⁻; or CH₂—COO⁻. In some embodiments, the CEX medium comprises asulfoethyl ligand.

In some embodiments, the CEX medium comprises a binding capacity ofbetween about 120 and about 160 mg lysozyme/ml resin.

In some embodiments, the recombinant polypeptide is produced by aeukaryotic host cell. In specific embodiments, the eukaryotic host cellis a mammalian host cell.

In some embodiments, the contacting is performed at a manufacturingscale.

In some embodiments, the composition further comprises at least oneimpurity. Examples of impurities include a DNA, RNA, lipid or protein.In some embodiments, the impurity comprises a protein. Examples of theprotein impurities include a truncated form of the recombinantpolypeptide, an aggregated form of the recombinant polypeptide, or amisfolded form of the recombinant polypeptide.

In some embodiments, the method provides a final composition comprisingthe recombinant polypeptides that do not bind the CEX medium, whereinthe final composition comprises less impurities than the compositionthat comprised the initial population of polypeptides.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 illustrates a SE Hicap Chromatogram for the enrichment ofsialylated etanercept. The load ratio was 31 mg/ml, the load pH was 5.6,and the load conductivity was 10 mS/cm. The product yield was 67%, andthe total sialic acid of the product was 15 moles of sialic acid/mole ofprotein. Peak 1 is composed of clipped etanercept. Peak 2 is composed ofnative etanercept. Peak 3 is composed of misfolded etanercept.

FIG. 2 illustrates a comparison of the TSA enrichment of etanercept byvarious CEX resins (SE Hicap, SP Sepharose, SP Sepharose XL, Nuvia S,Eshmuno, GigaCap S, and GigaCap CM). The results were normalized to 50%product yield.

FIG. 3 illustrates a comparison of the impurities removed from acomposition comprising etanercept by various CEX resins (SE Hicap, SPSepharose, SP Sepharose XL, Nuvia S, Eshmuno, GigaCap S, and GigaCapCM). The results were normalized to 50% product yield.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of enriching the level ofpost-translational modifications of recombinant polypeptides through theuse of a chromatography medium. In preferred embodiments, thechromatography medium is an ion exchange chromatography medium utilizedin a flow-through mode of operation.

In order to provide a clear understanding of the specification andclaims, the following definitions are provided below.

DEFINITIONS

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” can modify a numerical value above and below the stated value bya variance of, e.g., 10 percent, up or down (higher or lower).

As used herein, the term “protein” or “polypeptide” refers to a polymerof two or more of the natural amino acids or non-natural amino acids.

Polypeptides may be either monomers or multimers. For example, in oneembodiment, a protein of the invention is a dimer. A dimeric polypeptideof the invention may comprise two polypeptide chains or may consist ofone polypeptide chain (e.g., in the case of a scFc molecule). In oneembodiment, the dimers of the invention are homodimers, comprising twoidentical monomeric subunits or polypeptides (e.g., two identical Fcmoieties or two identical biologically active moieties). In anotherembodiment, the dimers of the invention are heterodimers, comprising twonon-identical monomeric subunits or polypeptides (e.g., comprising twodifferent clotting factors or portions thereof or one clotting factoronly). See, e.g., U.S. Pat. No. 7,404,956, incorporated herein byreference in its entirety.

“Recombinantly expressed polypeptide” and “recombinant polypeptide”refer to a polypeptide expressed from a host cell that has beengenetically engineered to express that polypeptide. The recombinantlyexpressed polypeptide can be identical or similar to polypeptides thatare normally expressed in the mammalian host cell. The recombinantlyexpressed polypeptide can also foreign to the host cell, i.e.heterologous to peptides normally expressed in the mammalian host cell.Alternatively, the recombinantly expressed polypeptide can be chimericin that portions of the polypeptide contain amino acid sequences thatare identical or similar to polypeptides normally expressed in themammalian host cell, while other portions are foreign to the host cell.Any polypeptide that is expressible in a host cell may be produced inaccordance with the present invention. The polypeptide may be expressedfrom a gene that is endogenous to the host cell, or from a gene that isintroduced into the host cell through genetic engineering. Thepolypeptide may be one that occurs in nature, or may alternatively havea sequence that was engineered or selected by the hand of man. Anengineered polypeptide may be assembled from other polypeptide segmentsthat individually occur in nature, or may include one or more segmentsthat are not naturally occurring. Polypeptides that may desirably beexpressed in accordance with the present invention will often beselected on the basis of an interesting biological or chemical activity.For example, the present invention may be employed to express anypharmaceutically or commercially relevant enzyme, receptor, antibody,hormone, regulatory factor, antigen, binding agent, etc. . . . .

Polypeptides that are “variants” of another peptide may have one or moremutations relative to the starting polypeptide, e.g., one or more aminoacid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions. In one embodiment, the polypeptide comprises an amino acidsequence which is not naturally occurring. Such variants necessarilyhave less than 100% sequence identity or similarity with the startingpolypeptide. In another embodiment, the variant will have an amino acidsequence from about 75% to less than 100% amino acid sequence identityor similarity with the amino acid sequence of the starting polypeptide,for example, from about 80% to less than 100%, from about 85% to lessthan 100%, from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%) and from about 95% to less than 100%, e.g.,over the length of the variant molecule. In one embodiment, there is oneamino acid difference between a starting polypeptide sequence and thesequence derived therefrom. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e. same residue) with thestarting amino acid residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity.

A “fusion” or “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences which normally exist inseparate proteins can be brought together in the fusion polypeptide, orthe amino acid sequences which normally exist in the same protein can beplaced in a new arrangement in the fusion polypeptide, e.g., fusion of aFactor VIII domain of the invention with an Ig Fc domain. A fusionprotein is created, for example, by chemical synthesis, or by creatingand translating a polynucleotide in which the peptide regions areencoded in the desired relationship. A chimeric protein can furthercomprises a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.

The term “aggregates”, as used herein, refers to polypeptide aggregates.It encompasses multimers (such as dimers, tetramers or higher orderaggregates) of the recombinant polypeptide to be purified and mayresult, e.g., in high molecular weight aggregates.

The term “chromatography” refers to any kind of technique whichseparates a recombinant polypeptide of interest from other moleculespresent in a mixture. Usually the recombinant polypeptide of interest isseparated from other molecules as a result of differences in bindingaffinity.

The term “binding” a molecule to a chromatography resin means exposingthe molecule to chromatography resin under appropriate conditions (e.g.pH/conductivity) such that the molecule is reversibly immobilized in oron the chromatography resin by virtue of ligand-protein interactions.Non-limiting examples include ionic interactions between the moleculeand a charged group or charged groups of the ion exchange material andan immunoglobulin.

The terms “flow-through,” “flow-through process,” “flow-through mode,”and “flow-through chromatography,” as used interchangeably herein, referto a product separation technique in which at least one product (e.g., arecombinant polypeptide with specific post-translational modifications)contained in a sample is intended to flow-through a chromatographicresin or media, while at least one potential contaminant or impuritybinds to the chromatographic resin or media.

The terms “chromatography resin”, “chromatography media” and“chromatography medium” are used interchangeably and refer to any kindof solid phase which separates a recombinant polypeptide of interestfrom other molecules present in a mixture. Usually, the recombinantpolypeptide of interest is separated from other molecules as a result ofdifferences in binding affinity between the other molecules in themixture and the recombinant polypeptide of interest. use of any known,or subsequently disclosed or developed, chromatography media or matrix.Examples, without limitation, of such media comprise: ion exchangemedia; anion exchange media; cation exchange media; hydroxyapatitemedia; hydrophobic interaction chromatography media; antibody-affinitymedia (e.g., Protein-A or variants thereof); immunoglobulin Fc-regionaffinity media (e.g., Fc-receptor affinity media); and, ligand-affinitymedia; receptor-affinity media; and mixed-mode media.

As used herein the terms “ion-exchange” and “ion-exchangechromatography” are used to refer to a chromatographic process in whicha recombinant polypeptide of interest interacts or does not interactwith a charged compound linked (such as by covalent attachment) to asolid phase ion exchange material such that the recombinant polypeptideof interest interacts non-specifically with the charged compound more orless than other recombinant polypeptides or impurities in the mixture.The contaminant in the mixture elutes from a column of the ion exchangematerial faster or slower than the recombinant polypeptide of interestor are bound to or excluded from the resin relative to the recombinantpolypeptide of interest. “Ion-exchange chromatography” specificallyincludes cation exchange, anion exchange, and mixed mode ion exchangechromatography.

The “pI” or “isoelectric point” of a polypeptide refer to the pH atwhich the polypeptide's positive charge balances its negative charge, pIcan be calculated from the net charge of the amino acid residues orsialic acid residues of attached carbohydrates of the polypeptide or canbe determined by isoelectric focusing.

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (i.e., a cation exchange resin) or positively charged(i.e., an anion exchange resin). The charge may be provided by attachingone or more charged ligands to the solid phase, e.g., by covalentlinking. Alternatively, or in addition, the charge may be an inherentproperty of the solid phase (e.g., as is the case for silica, which hasan overall negative charge).

The phrase “anion exchange resin” or “AEX” refers to a solid phase whichis positively charged, e.g., having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia). Anion exchange chromatography canbind the target molecule (e.g., an Fc region containing target protein)followed by elution or can predominately bind the impurities while thetarget molecule “flows through” the column.

The phrase “cation exchange resin” or “CEX” refers to a solid phasewhich is negatively charged, and which thus has free cations forexchange with cations in an aqueous solution passed over or through thesolid phase. A negatively charged ligand attached to the solid phase toform the cation exchange resin may, e.g., be a carboxylate or sulfonate.For example, cation exchange chromatography can be performed underconditions in which the resin bind the target molecule (e.g., an Fcregion containing target protein) followed by elution (cation exchangebind and elution chromatography or “CIEX”). Alternatively, CEX can berun in a mode which it predominately binds the impurities while thetarget molecule “flows through” the column (cation exchange flow-throughchromatography). Commercially available CEX resins include Fractogel SEHicap, GE SPXL, GE SP Sepharose, Millipore Eshmuno S, Tosoh Gigacap CM,Tosoh Gigacap S-650, or BioRad Nuvia S. The phrase “cation exchangeresin” or “CEX” also refers to a mixed-mode resin that is partially orentirely operated in cation-exchange mode of chromatography.Commercially available mixed-mode resins include Capto MMC. Thepurification method disclosed herein utilizes a cation exchangechromatography step which is performed in a flow-through mode.

Chromatography Methods

The present invention utilizes a “flow-through” mode of operationwherein a recombinant polypeptide is allowed to contact a chromatographymedium (or other matrix). During the contacting, a recombinantpolypeptide having a selected characteristic (such as a particularpost-translational modification) preferentially does not bind to thechromatography medium (or other matrix) while recombinant polypeptides(as well as other impurities) not having the selected characteristic, orhaving less of the selected characteristic (such as a recombinantpolypeptide having a lower overall net negative charge or a lower sialicacid content) binds to the medium (or matrix). The flow-through,containing the enriched recombinant polypeptide, is recovered. Therecombinant polypeptide mixture obtained is enriched with a higherconcentration of product having the selected (target) characteristiccompared to the composition containing the initial population ofrecombinant polypeptides prior to contacting with the chromatographymedium.

In some embodiments, the present invention is directed to selectivelyenriching recombinant polypeptides wherein the selected or desiredproduct characteristic is that of having increased or enhanced overall(total) levels of sialic acid content. In some embodiments, arecombinant polypeptide with increased total sialic acid content isobtained by contacting a composition that contains a population ofexchange chromatography medium (e.g., SE Hicap) with a mixture of therecombinant polypeptide. Undesired products (e.g., product with a lowersialic acid content, and other impurities) are allowed to bind thechromatography medium, then the selected product (with high sialic acidcontent) flows through the column and is recovered.

Methods of the present invention can be adapted and applied to theseparation/purification of recombinant polypeptides based on any numberof physical, biological, and/or chemical characteristics. For example,product isoforms may be selectively separated on the basis of chargeand/or hydrophobicity by using appropriate adsorbents (such as, forexample, strong or weak cation exchange resins for charge basedseparations and hydrophobic adsorbents for separations based onhydrophobicity). Additionally, methods of the invention may be appliedusing mixed-mode chromatography (mixed-mode media) for separations basedon two orthogonal product attributes (e.g., charge and hydrophobicity).

In some embodiments, the invention relates to a method for enriching,increasing, enhancing, or augmenting the level of post-translationalmodification of recombinant polypeptides comprising: contacting acomposition which comprises an initial population of recombinantpolypeptides having different levels of post-translational modificationwith a cation exchange chromatography (CEX) medium operated in aflow-through mode; wherein recombinant polypeptides that do not bind theCEX medium are separated from recombinant polypeptides that bind the CEXmedium, and wherein the recombinant polypeptides that do not bind theCEX medium comprise a higher or increased level of post-translationalmodification compared to the bound recombinant polypeptides.

In some embodiments, the recombinant polypeptides that do not bind theCEX medium are more negatively charged than the polypeptides or otherimpurities that bind the CEX resin. In some embodiments, the inventionfurther comprises recovering the recombinant polypeptides that do notbind the CEX medium.

In some embodiments, recombinant polypeptides with higher levels ofspecific post-translational modifications are separated from recombinantpolypeptides with lower levels of post-translational modificationsthrough the use of a CEX medium operated in flow-through mode.“Separating” refers to increasing the degree of purity of a recombinantpolypeptide of interest from a composition or sample comprising thepolypeptide and one or more impurities or contaminants. In someembodiments, the recombinant polypeptide of interest is separated fromthe other polypeptides or impurities in the composition through the useof charge. In some embodiments, the recombinant polypeptide of interestis more negatively charged than other polypeptides or impurities in thecomposition such that the recombinant polypeptide of interest does notbind to the CEX medium operated in flow-through mode. In someembodiments, the degree of purity of the recombinant polypeptide ofinterest is increased by removing (completely or partially) at least oneimpurity from the composition. In some embodiments, thepost-translational modification is sialylation or gamma-carboxyglutamate(Gla) formation.

In some embodiments, the post-translational modification is sialylation.Sialylation is the final step of human glycosylation. Total sialicresidue refers to the total number of sialic residues on a givenrecombinant polypeptide. The sialylation of a protein is critical toprotein function, as the in vivo biological activity of glycosylatedproteins is known to be dependent on the number of sialic acid units permolecule. Shiestl, M., et al. Nature Biotechnology 29(4):310 (2011). Theloss of sialic acid frequency leads to reduced glycoprotein solubilityand reduced circulatory half-life. As a result the purification andtherapeutic effectiveness of a recombinant polypeptide is dependent onthe sialic acid content. The glycosylation profile of a polypeptide canbe affected by many factors including the type of production cell line,the growth conditions, and the polypeptide sequence. Shiestl, M., et al.Nature Biotechnology 29(4):310 (2011).

In some embodiments, the total sialic acid content of the recombinantpolypeptide is between about 0.5 and about 6, between about 1 and about4, or between about 1 and about 3 moles of sialic per mole of proteinhigher than that of the initial population of recombinant polypeptides.In some embodiments, the total sialic acid content of the recombinantpolypeptide is between about 0.6 and about 2.7 moles of sialic per moleof protein higher than that of the initial population of recombinantpolypeptides. In some embodiments, the total sialic acid content of therecombinant proteins is at least about 0.5, at least about 1.0, at leastabout 1.5, at least about 2.0, at least about 2.5, at least about 3.0,at least about 3.5, at least about 4.0, at least about 4.5, at leastabout 5.0, at least about 5.5, or at least about 6.0 moles of sialic permole of protein higher than that of the initial population ofrecombinant polypeptides.

In some embodiments, the total sialic content of the recombinantpolypeptides that do not bind the CEX medium is between about 5% andabout 100%, between about 5% and about 90%, between about 5% and about80%, between about 5% and about 70%, between about 5% and about 60%,between about 5% and about 50%, and between about 5% and about 40%higher than that of the composition containing the initial population ofrecombinant polypeptides. In some embodiments, the total sialic contentof the recombinant polypeptides that do not bind the CEX medium is atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99% or at least about 100%higher than that of the composition containing the initial population ofrecombinant polypeptides.

In some embodiments, the total sialic acid content of the recombinantpolypeptides that do not bind the CEX medium is between about 13 andabout 20, between about 13.5 and about 19, between about 14 and about18, between about 14 and about 17, or between about 14 and about 16.5moles of sialic acid per mole of protein. In some embodiments, the totalsialic acid content of the recombinant polypeptides that do not bind theCEX medium is between about 14.1 and about 16.2.

In some embodiments, the total sialic content of the initial content ofthe initial population of recombinant polypeptides is between about 10and about 14, between about 10.5 and about 14, between about 11 andabout 14, between about 11.5 and about 14, between about 12 and about14, between about 12.5 and about 14, or between about 13 and about 14moles of sialic acid per mole of protein. In some embodiments, the totalsialic acid content of the initial population of recombinantpolypeptides is at least about 10, at least about 10.5, at least about11, at least about 11.5, at least about 12, at least about 12.5, atleast about 13, at least about 13.5 or at least about 14 moles of sialicacid per mole of protein. In some embodiments, the total sialic acidcontent of the initial population of recombinant polypeptides is atleast about 13.5 moles of sialic acid per mole of protein.

In some embodiments, the post-translational modification isgamma-carboxyglutamate (Gla) formation. Thecarboxylation/gamma-carboxyglutamic (GLA) domain is a Vitamin-Kdependent domain. At the GLA domain, Vitamin K mediates thecarboxylation of glutamate residues to form gamma-carboxyglutamate(Gla). Vermeer, C. Biochem J. 266: 625-636 (1990). Gla plays a key rolein calcium binding, and has been found to be critical for specificconformational transitions in clotting factor proteins. Freedman, S. J.et al., J. Biol. Chem. 271(27): 16227-36 (1996).

In some embodiments, the Gla content of the recombinant polypeptidesthat do not bind the CEX medium is between about 5% and about 100%,between about 5% and about 90%, between about 5% and about 80%, betweenabout 5% and about 70%, between about 5% and about 60%, between about 5%and about 50%, and between about 5% and about 40% higher than that ofthe initial population of recombinant polypeptides.

In some embodiments, the total Gla content of the recombinantpolypeptides that do not bind the CEX medium is between about 5% andabout 100%, between about 5% and about 90%, between about 5% and about80%, between about 5% and about 70%, between about 5% and about 60%,between about 5% and about 50%, and between about 5% and about 40%higher than that of the initial population of recombinant polypeptides.

In some embodiments of the invention, a chromatography medium in“flow-through” mode of operation is utilized to enrich a composition ofrecombinant polypeptides having a specific post-translationalmodification.

The load ratio describes the total amount of the composition that iscontacting the chromatography medium per volume of medium. If the loadratio is too high, then, in the case of a column run in flow-throughmode, some impurities may not bind the medium. In some embodiments, thecontacting of the composition with the medium occurs at a load ratio ofat least about 30 mg total protein/ml CEX medium, at least about 35 mgtotal protein/ml CEX medium, at least about 40 mg total protein/ml CEXmedium, at least about 45 mg total protein/ml CEX medium, at least about50 mg total protein/ml CEX medium, at least about 55 mg total protein/mlCEX medium, at least about 60 mg total protein/ml CEX medium, at leastabout 70 mg total protein/ml CEX medium, at least about 80 mg totalprotein/ml CEX medium, at least about 90 mg total protein/ml CEX medium,at least about 100 mg total protein/ml CEX medium, at least about 110 mgtotal protein/ml CEX medium, at least about 120 mg total protein/ml CEXmedium, at least about 130 mg total protein/ml CEX medium, at leastabout 140 mg total protein/ml CEX medium, or at least about 150 mg totalprotein/ml CEX medium. In some embodiments, the contacting occurs at aload ratio of 41 mg total protein/ml CEX medium. In some embodiments,the contacting of the composition with the medium occurs at a load ratioof between about 30 mg total protein/ml CEX medium and about 150 mgtotal protein/ml CEX medium, between about 30 mg total protein/ml CEXmedium and 125 mg total protein/ml CEX medium, between about 30 mg totalprotein/ml CEX medium and 100 mg total protein/ml CEX medium, betweenabout 30 mg total protein/ml CEX medium and 75 mg total protein/ml CEXmedium, between about 30 mg total protein/ml CEX medium and 60 mg totalprotein/ml CEX medium, or between about 33 mg total protein/ml CEXmedium and 54 mg total protein/ml CEX medium.

The pH of the contacting step is critical because it determines whichcharged molecules will bind the column and which will not bind. In someembodiments, the contacting of the composition with the medium occurs ata pH of at least about 3, at least about 4, at least about 5, at leastabout 5.5, at least about 6, at least about 6.5, at least about 7, atleast about 8 or at least about 9. In some embodiments, the contactingoccurs at a pH of 5.6. In some embodiments, the contacting of thecomposition with the medium occurs at a pH between about 3 and about 9,between about 4 and about 8, between about 4 and about 7, between about5 and about 7, between about 5 and about 6, or between about 5.5 andabout 5.8.

The conductivity of the contacting step is critical because itdetermines which molecules will bind the column and which will not bind.In some embodiments, the contacting of the composition with the mediumoccurs at a conductivity of at least about 8 mS/cm, at least about 8.5mS/cm, at least about 9 mS/cm, at least about 10 mS/cm, at least about10.5 mS/cm, at least about 11 mS/cm, at least about 11.5 mS/cm, at leastabout 12 mS/cm, at least about 12.5 mS/cm, or at least about 13 mS/cm.In some embodiments the contacting occurs at a conductivity of 10 mS/cm.In some embodiments the contacting occurs at a conductivity of betweenabout 8 mS/cm and about 12 mS/cm, between about 8.5 mS/cm and 11.5mS/cm, between about 9 mS/cm and about 11.5 mS/cm, between about 9 mS/cmand about 11 mS/cm, or between about 9.5 mS/cm and 11 mS/cm.

In some embodiments, the chromatography medium is ion exchange media;anion exchange media; cation exchange media; hydroxyapatite media;hydrophobic interaction chromatography media; antibody-affinity media(e.g., Protein-A or variants thereof); immunoglobulin Fc-region affinitymedia (e.g., Fc-receptor affinity media); and, ligand-affinity media;receptor-affinity media; or mixed-mode media. In preferred embodiments,the chromatography medium is a CEX resin. Embodiments of the inventioninclude use of any known, or subsequently disclosed or developed, CEXchromatography media or matrix. In some embodiments, the CEX resin is aFractogel SE Hicap, GE SPXL, GE SP Sepharose, Millipore Eshmuno S, TosohGigacap CM, Tosoh Gigacap S-650, BioRad Nuvia S, or Capto MMC. In someembodiments, the CEX medium comprises a ligand that is sulfoethyl;sulphopropyl; sulfopropyl; CH₂—SO₃; CH₂CH₂CH₂SO₃ ⁻; SO₃ ⁻; CH₂—COO⁻; ora multimodal weak cation exchanger. In some embodiments, the CEX mediumcomprises a matrix that is: crosslinked polymethacrylate, 6%cross-linked agarose with dextran surface extenders, agarose, surfacegrafted rigid polyvinyl ether hydrophilic polymer, or highlycross-linked agarose. In some embodiments, the CEX medium is any knownmatrix. In some embodiments, the CEX medium comprises a sulfoethylligand. In some embodiments, the CEX resin is Fractogel SE Hicap.

In some embodiments, the CEX medium comprises a binding capacity orligand capacity of between about 100 and about 200 mg lysozyme/ml resin,between about 110 and 190 mg lysozyme/ml resin, between about 120 andabout 180 mg lysozyme/ml resin, between about 120 and about 170 mglysozyme/ml resin, between about 120 and about 160 mg lysozyme/ml resin,or between about 125 and 160 mg lysozyme/ml resin.

The ion exchange rate between the medium and the composition increasesas the temperature increases. Temperature can also affect theselectivity of the column. In some embodiments, the temperature duringthe contacting of the composition with the medium is between about 16and about 26° C., between about 18 and about 24° C., or between about 20and about 23° C. In some embodiments, the temperature during thecontacting of the composition with the medium is at least about 15° C.,at least about 16° C., at least about 17° C., at least about 18° C., atleast about 19° C., at least about 20° C., at least about 21° C., atleast about 22° C., at least about 23° C., at least about 24° C., atleast about 25° C., at least about 26° C. or at least about 30° C. Insome embodiments, the temperature during the contacting of thecomposition with the medium is about 22° C.

A higher flow rate decreases the process time, but a higher flow ratemay decrease the efficiency of separation, due to decreased contactingtime between the composition and the medium. In some embodiments, theprocess flow rate is between about 75 cm/hr and about 150 cm/hr, betweenabout 100 cm/hr and about 150 cm/hr, or between about 120 cm/hr andabout 150 cm/hr. In some embodiments, the process flow rate is at leastabout 75 cm/hr, at least about 80 cm/hr, at least about 90 cm/hr, atleast about 100 cm/hr, at least about 110 cm/hr, at least about 115cm/hr, at least about 120 cm/hr, at least about 125 cm/hr, at leastabout 130 cm/hr, at least about 135 cm/hr, at least about 140 cm/hr, orat least about 150 cm/hr. In some embodiments, the process flow rate isabout 125 cm/hr.

Embodiments of the invention include methods for recovering a selectedrecombinant polypeptide at a manufacturing scale; including a methodwherein the selected recombinant polypeptide is a therapeutically usefulor beneficial compound.

In some embodiments, the composition containing the initial populationof recombinant polypeptides contains only the recombinant polypeptide ofinterest with differing levels of the selected characteristic. In someembodiments, the composition containing the initial population ofrecombinant polypeptides also contains additional polypeptides that havea different amino acid sequence than the recombinant polypeptide ofinterest. In some embodiments, the additional polypeptides arerecombinant. In some embodiments, the composition containing the initialpopulation of recombinant polypeptides also contains impurities. In someembodiments, the impurity is a DNA, RNA, lipid or protein molecule. Insome embodiments, the impurity is a truncated form of the recombinantpolypeptide, an aggregated form of the recombinant polypeptide, or amisfolded form of the recombinant polypeptide. In some embodiments, thecomposition containing the recombinant polypeptide that does not bindthe CEX medium has less impurities that the composition that containedthe initial population of recombinant polypeptides. In some embodiments,the composition containing the recombinant polypeptide that does notbind the CEX medium has at least about 5%, at least about 10%, at leastabout 25%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or at leastabout 99% less impurities than the composition that contained theinitial population of recombinant polypeptides.

In some embodiments, the recombinant polypeptides that do not bind theCEX medium are between about 10% and about 90%, between about 20% andabout 80%, between about 30% and about 80%, between about 40% and about80%, between about 50% and about 80%, or between about 55% and about 80%of the recombinant polypeptides of the composition comprising theinitial population. In some embodiments, the recombinant polypeptidesthat do not bind the CEX medium are between about 55% and about 80% ofthe recombinant polypeptides of the composition comprising the initialpopulation.

In one embodiment, product may be selectively enriched/separated, forexample, based on peak product pI values wherein, for example, higher pIisoforms may be separated from lower pI product isoforms on a cationexchange adsorbent.

In some embodiments, the recombinant polypeptides that do not bind theCEX medium are subjected to one or more further purification stepsfollowing the CEX chromatography. In some embodiments, the compositioncontaining the initial population of recombinant polypeptides issubjected to one or more purification steps prior to the contacting withthe CEX medium. In some embodiments, the pH and the conductivity of thecomposition with the initial population of recombinant polypeptides areadjusted prior to contacting the composition with the CEX medium. Insome embodiments the pH of the recombinant polypeptides that do not bindthe CEX medium is adjusted following the contacting with the CEX medium.

Recombinant Polypeptides of the Invention

Embodiments of the invention are useful for obtaining highly homogeneousmixtures of a wide variety of recombinant polypeptides with specificlevels of post-translational modifications. Some examples of suchrecombinant polypeptides include, without limitation, proteins andprotein fragments (i.e., full-length and partial lengthpolypeptides/peptides), antibodies (immunoglobulins), heterologousfusion proteins, etc. . . . . In some embodiments, the polypeptide is adimer. In some embodiments, the polypeptide is a monomer.

In one embodiment, the recombinant polypeptide is a non-immunoglobulinproteins (or fragments thereof) fused with an immunoglobulin (ordomains, regions, of fragments thereof). In some embodiments, therecombinant polypeptide comprises an Fc domain. In some embodiments, therecombinant polypeptide comprises an antibody. In one embodiment, forexample, methods of the invention are used for separation/purificationof a recombinant polypeptide comprising an extracellular receptorligand-binding domain linked (i.e., “fused”) with the Fc-region of animmunoglobulin (such as the Fc region of an IgG molecule). Examples ofFc-fusion proteins can be seen in Table 1.

TABLE 1 Key Fc-fusion proteins and monoclonal antibodies (mAbs) in theclinic Trade name Indication of first (generic name) Description FDAapproval Stage Company Fc-fusion Nulojix CTLA-4 fused to the Fc of Organrejection FDA Approved Bristol-Meyers (belatacept) human IgG1 (2011)Squibb Eylea VEGFR1/VEGFR2 fused Age related macular FDA ApprovedRegeneron (aflibercept) to the Fc of human IgG1 degeneration (2011)Pharmaceuticals Arcalyst IL-1R fused to the Fc of Cryopyrin-associatedFDA Approved Regeneron (rilonacept) human IgG1 periodic syndromes (2008)Pharmaceuticals NPlate Thrombopoietin-binding Thrombocytopenia in FDAApproved Amgen/Pfizer (romiplostim) peptide fused to the Fc of chronicimmune (2008) human IgG1 thrombocytopenic purpura patients OrenciaMutated CTLA-4 fused to Rheumatoid arthritis FDA Approved Bristol-Meyers(abatacept) the Fc of human IgG1 (2005) Squib Amevive LFA-3 fused to theFc of Psoriasis and FDA Approved Astellas Pharma (alefacept) human IgG1transplant rejection (2003) Enbrel TNFR fused to the Fc of Rheumatoidarthritis FDA Approved Amgen/Pfizer (etanercept) human IgG1 (1998) mAbsRituxan/MabThera Chimeric mouse/human B cell lymphomas FDA ApprovedBiogen (rituximab) IgG1 targeting CD20 (2006) Idec/Genentech Hoffman-LaRoche (Europe) Herceptin Chimeric mouse/human Breast cancer and FDAApproved Genentech (trastuzumab) IgG1 targeting HER2 gastroesophageal(2006) junction adenocarcinoma Campath/Lemtrada Humanized IgG1 targetingB cell chronic FDA Approved Genzyme (alemtuzumab) CD52 on B and Tlymphocytic leukemia. (2007) lymphocytes In phase IIIa trials formultiple sclerosis Prolia/Xgeva Fully human IgG2 Osteoporosis FDAApproved Amgen (denosumab) targeting RANKL (2010) Tysabri Humanized IgG4tageting Multiple sclerosis and FDA Approved Biogen Idec and(natalizumab) alpha-4 integrin Crohn's disease (2004) Élan VectibixFully human IgG2 Metastatic colorectal FDA Approved Amgen (panitumumab)targeting EGFR, ErbB-1 cancer (in patients with (2006) and HER1non-mutated KRAS Soliris Humanized IgG2/4κ Paroxysmal nocturnal FDAApproved Alexion (eculizumab) targeting complement haemoglobinuria to(2007) Pharmaceuticals protein C5 reduce haemolysis Erbitux Chimericmouse human Metastatic colorectal FDA Approved Bristol-Myers (cetuximab)IgG1 targeting EGFR, cancer (in patients with (2006) Squibb and EliErbB-1 and HER1 non-mutated KRAS Lilly Avastin Humanized IgG1 targetingMetastatic colorectal FDA Approved Genentech/Roche (bevacizumab) VEGFcancer and HER2- (2008) negative metastatic Withdrawn breast cancer(2011) Remicade Chimeric mouse human Psoriasis, Crohn's FDA ApprovedJanssen (infliximab) IgG1 targeting TNF-α disease, ankylosing (1998)Biotech/Schering- spondylitis, Plough rheumatoid arthritis

In one embodiment, the recombinant polypeptide is an Fc fusionpolypeptide comprising a ligand binding domain of a receptor. In someembodiments, the receptor is a Tumor necrosis factor (“TNF”) receptor.In some embodiments, the recombinant polypeptide is etanercept.Etanercept is a dimeric recombinant therapeutic glycoprotein, whichconsisting of the extracellular ligand binding portion of the human 75kilodalton human tumor necrosis factor receptor linked to the constantregion (Fc) of human IgG1. The TNF receptor region binds to solubleTNF-α found in the bloodstream, thereby reducing a variety ofinflammatory responses, including many autoimmune diseases. Etanerceptis as a TNF inhibitor, an acts as a decoy receptor that binds to TNF.Zalevsky, J. et al., J. Immunol. 179(3): 1872-83. Etanercept acts in asimilar function to naturally occurring soluble TNF receptors. However,as etanercept is a fusion protein, it has a greater half-life in thebloodstream, and therefore has a longer impact than a naturallyoccurring receptor. Madhusudan, S. J. Clin Oncol. 23(25): 5950-9. The Fcportion of the fusion protein transiently anchors to Fc receptorsexpressed on the surface of endothelial cells, which delays thedegradation and increases the half-life of etanercept.

Etanercept requires N-glycosylation for biological activity.Glycosylated proteins are complex molecules and even a well-controlledproduct may consist of several hundred or more glycoforms with differentglycan compositions on the same amino acid sequence. The in vivobiological activity of glycosylated proteins is known to be dependent onthe number of sialic acid units per molecule, which is a result of theavailable sialylation sites, the antenniarity of the N-glycans and thecompleteness of sialylation. Shiestl, M., et al. Nature Biotechnology29(4):310 (2011).

In some embodiments, the recombinant polypeptide comprises a clottingfactor. In some embodiments, the clotting factor is selected from FactorVII (FVII), FVIIa, Factor VIII (FVIII), Factor IX (FIX), or FIXa (FIX).In some embodiments, the FVIII is full-length FVIII or B-domain deletedFVIII. In some embodiments, the FVIII is single chain FVIII or dualchain FVIII.

In some embodiments, the recombinant polypeptide is a monomer-dimerhybrid. A monomer-dimer hybrid is a chimeric protein having a dimericaspect and a monomeric aspect, wherein the dimeric aspect relates to thefact that it is comprised of two polypeptide chains each comprised of aportion of an immunoglobulin constant region, and wherein the monomericaspect relates to the fact that only one of the two chains is comprisedof a therapeutic biologically active molecule. Monomer-dimer hybrids aredescribed in detail is U.S. Pat. No. 7,404,956, which is incorporatedherein by reference in its entirety.

“Factor VII” or “FVII” refers to a coagulation factor proteinsynthesized in the liver and secreted into the blood as a single chainzymogen with a molecular weight of approximately 50 kDa. The FVIIzymogen is converted into an activated form (FVIIa) by proteolyticcleavage. FVII is disclosed in U.S. Publ. No. 2011/0046061 and Int'lPubl. No. PCT/US2013/44842, each of which is incorporated herein byreference in its entirety.

“Factor VIII” or “FVIII” refers to a blood coagulation factor proteinand species and sequence variants thereof that includes, but is notlimited to, the 2351 amino acid single-chain precursor protein (with a19-amino acid hydrophobic signal peptide), the mature 2332 amino acidfactor VIII protein of approximately 270-330 kDa with the domainstructure A1-A2-B-A3-C1-C2, as well as the circulating heterodimer oftwo chains that form as a result of proteolytic cleavage after R1648 ofa heavy chain form composed of A1-A2-B (in the range of 90-220 kD) ofamino acids 1-1648 (numbered relative to the mature FVIII form) and alight chain A3-C1-C2 of 80 kDa of amino acids 1649-2232, each of whichis depicted schematically in FIG. 1. “Factor VIII” or “FVIII” also canbe sequence variants that retain at least a portion of the biologicalactivity of the native circulating protein, including truncatedsequences, a sequence that includes heterologous amino acids, or asingle chain FVIII (scFVIII) in which the heavy and light chains arecovalently connected by a linker. As used herein, “FVIII” shall be anyfunctional form of factor VIII molecule with the typical characteristicsof blood coagulation factor VIII capable of in vivo or in vitrocorrection of human factor VIII deficiencies (e.g., hemophilia A). FVIIIor sequence variants have been isolated, characterized, and cloned, asdescribed in U.S. Pat. or Publ. Nos. U.S. Pat. Nos. 4,757,006;7,138,505; 5,004,804; 5,198,349, 5,250,421; 5,919,766; 2010/0081615;2013/0017997 and 2013/0108629 each of which is incorporated herein byreference in its entirety.

“B domain” of Factor VIII, as used herein, is the same as the B domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage by thrombin, e.g., residuesSer741-Arg1648 of full length human factor VIII. The other human factorVIII domains are defined by the following amino acid residues: A1,residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residuesSer1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the factor VIII light chain activation peptide.The locations of the boundaries for all of the domains, including the Bdomains, for porcine, mouse and canine factor VIII are also known in theart. Preferably, the B domain of Factor VIII is deleted (“B domaindeleted factor VIII” or “BDD FVIII”). An example of a BDD FVIII isREFACTO (recombinant BDD FVIII). The B domain of FVIII is discussed inU.S. Publ. No. 2013/0108629, which is incorporated herein by referencein its entirety.

A “B domain deleted factor VIII” may have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted factor VIII sequence of the presentinvention comprises any one of the deletions disclosed at col. 4, line 4to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also inU.S. Pat. No. 6,346,513). In some embodiments, a B domain deleted factorVIII of the present invention has a deletion disclosed at col. 2, lines26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No.6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). Insome embodiments, a B domain deleted factor VIII has a deletiondescribed in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No.5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720;col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col.5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, andexample 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635;or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments,a B domain deleted factor VIII has a deletion of most of the B domain,but still contains amino-terminal sequences of the B domain that areessential for in vivo proteolytic processing of the primary translationproduct into two polypeptide chain, as disclosed in WO 91/09122, whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted factor VIII is constructed with adeletion of amino acids 747-1638, i.e., virtually a complete deletion ofthe B domain. Hoeben R. C., et al, J. Biol. Chem. 265 (13): 7318-7323(1990), incorporated herein by reference in its entirety. A. B domaindeleted factor VIII may also contain a deletion of amino acids 771-1666or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng.2(4): 301-6 (1988), incorporated herein by reference in its entirety.Additional B domain deletions that are part of the invention include,e.g.: deletion of amino acids 982 through 1562 or 760 through 1639(Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741through 1646 (Kaufman (PCT published application No. WO 87/04187)),747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648(Pasek (PCT application No. 88/00831)), 816 through 1598 or 741 through1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), eachof which is incorporated herein by reference in its entirety. Each ofthe foregoing deletions may be made in any Factor VIII sequence. Bdomain deletions of FVIII are disclosed in U.S. Publ. No. 2013/0108629,which is incorporated herein by reference in its entirety.

“Factor IX” and “FIX,” as used herein, means functional Factor IXpolypeptide in its normal role in coagulation, unless otherwisespecified. Thus, the term Factor IX includes variant polypeptides thatare functional and the polynucleotides that encode such functionalvariant polypeptides. Preferred Factor IX polypeptides are the human,bovine, porcine, canine, feline, and murine Factor IX polypeptides. Thefull length polypeptide and polynucleotide sequences of Factor IX areknown, as are many functional variants, e.g., fragments, mutants andmodified versions. Factor IX polypeptides include full-length Factor IX,full-length Factor IX minus Met at the N-terminus, full-length Factor IXminus the signal sequence, mature Factor IX (minus the signal sequenceand propeptide), and mature Factor IX with an additional Met at theN-terminus. Factor IX is preferably made by recombinant means(“recombinant Factor IX” or “rFIX”), i.e., it is not naturally occurringor derived from plasma. FIX is disclosed in U.S. Publ. Nos. 2011/0046060and 2013/0202595, each of which is incorporated herein by reference inits entirety.

In some embodiments, the recombinant polypeptide is an antibody. In someembodiments, the recombinant polypeptide is a monoclonal antibody. Insome embodiments, the antibody is a humanized antibody. In someembodiments, the recombinant polypeptide is a chimeric antibody. In someembodiments, the recombinant polypeptide is any antibody disclosed inU.S. Pat. No. 7,300,773, which is incorporated herein by reference inits entirety.

In some embodiments, the recombinant polypeptide is a receptor. In someembodiments, the antibody is a receptor tyrosine kinase. In someembodiments, the receptor is any receptor disclosed in U.S. Pat. No.7,300,773, which is incorporated herein by reference in its entirety.

In some embodiments the recombinant polypeptide is a growth factor orother signaling molecule. In some embodiments the recombinantpolypeptide is a G-protein coupled receptor. In some embodiments therecombinant polypeptide is any polypeptide disclosed in U.S. Pat. No.7,300,773.

In some embodiments, the recombinant polypeptide is produced by a hostcell. In some embodiments, the recombinant polypeptide is produced by aeukaryotic host cell. In some embodiments, the recombinant polypeptideis produced by a mammalian host cell.

ABBREVIATIONS

AEX: anion exchange chromatography

CEX: cation exchange chromatography

CV: column volume

DF: diafiltration

DoE: design of experiments

HIC: hydrophobic interaction chromatography

HMW: high molecular weight aggregates

LMW: low molecular weight aggregates

HCP: host cell protein

Peak 1: clipped etanercept

Peak 2: native etanercept

Peak 3: misfolded etanercept

LRV: Log₁₀ (total impurity in load/total impurity in eluate)

CV: column volume

PS: pilot scale

OD: optical density

TSA: total sialic acid

UF: ultrafiltration

WFI: water for injection

By applying knowledge within the skill of those in the art, embodimentsof the invention may be modified and/or adapted for variousapplications, without undue experimentation, without departing from thegeneral concept of the present invention. Therefore, such adaptationsand modifications are intended to be within the meaning and range ofequivalents of the disclosed embodiments, based on the teaching andguidance presented herein.

EXAMPLES Example 1 Enrichment of TSA and Removal of Impurities ThroughUse of a SE Hicap Column Summary

A chromatography process was developed for theisolation/purification/enrichment of recombinant polypeptides withenhanced or increased levels of sialylation compared to recombinantpolypeptides with decreased or lower levels of sialylation present inthe same initial mixture.

The recombinant polypeptide utilized in this example is the TNFR-Fcfusion protein etanercept. Highly sialylated forms of recombinantpolypeptides can represent a highly desirable class of therapeuticallyadvantageous protein isoforms. The present example describes developmentof a robust process capable of enriching the content ofrecovered/isolated, highly sialylated forms of recombinant polypeptideswhile maintaining acceptable product recovery and yield. In thisparticular example, a process was developed wherein SE Hicapchromatography was used to recover/isolate highly sialylated forms of anFc-fusion protein.

SE Hicap utilized in flow-through mode was selected primarily for itsTSA enrichment potential, ability to reduce Peak 3 and aggregates,capability for robust DNA clearance, and ease of interface with theother process steps. SE Hicap enriches TSA by electrostaticallyrepelling the negatively-charged sialylated etanercept into theflow-through, while retaining the more positively-charged (lessersialylated) protein when run at a pH below its pI. It was found thatload pH, load conductivity, and loading ratio are all key and criticalparameters for this column. Process temperature, process flow rate, andresin ligand capacity had negligible impact on yield and productattributes under the conditions studied and were judged to be non-keyprocess parameters.

Materials

Solutions: The pH and conductivity of each buffer were verified beforeuse.

Load Material and Adsorbent: All samples were frozen at −70° C. andthawed at ambient temperature prior to initiating chromatography. Forall of the experiments, the load pH was adjusted with 1 M acetic acid, 1M citric acid, or 2.4 M Tris base. Then, the load conductivity wasadjusted with 1 M NaCl in acetate or citrate buffer. RO/DI water wasadded if the load conductivity was too high.

Chromatography Method

All columns were packed with a 20% compression factor to a packed bedheight of 15 cm or 16 cm. The chromatography steps were carried out withan AKTA Explorer 900 (GE Healthcare Life Sciences) and the Unicorninterface software (version 5.11) at approximately 22° C. in a down-flowmode. Throughout all of the development studies operated in flow-throughmode, the chromatography method generally consisted of an equilibrationstep, a load step, a wash step to remove non-bound protein, a strip stepto remove impurities for analysis, a cleaning step with 1 N NaOH, and astorage step in 0.1 N NaOH. The cleaning and storage conditions for thisstep were not studied; rather these follow the manufacturer'srecommendations. The wash fractions were pooled with the flow-throughfractions unless otherwise noted. The flow-through, wash, and stripfractions were analyzed for yield and product attributes.

Resin Screening, Column Interfacing, and Buffer Matrix Method

Static binding capacity calculations were used to screen candidate CEXresins for their ability to bind protein. This was determined by bufferexchanging the candidate resins with equilibration buffer and thenincubating the composition containing the Etanercept overnight with aknown volume of candidate resins on a shaker. The mixtures of resin andprotein were spun down and the supernatants were subsequently measuredfor A280. The resins with high static capacities were then packed incolumns and further evaluated in bind/elute or flow-through mode.Product yield, aggregates, Peak 1, Peak 2, Peak 3, TSA, HCP, and DNAwere measured to gauge separation performance. Resins with highcapacities that had poor separation of product-related impurities duringelution were further evaluated as a capture step, but eliminated as apolishing step. However, resins with low capacities that had goodseparation of impurities were further evaluated in flow-through mode, aswas the case with SE Hicap.

SE Hicap was selected specifically for its ability to enrich TSA andremove aggregates, DNA, and some Peak 3 and HCP. In addition, SE Hicapin flow-through mode had no challenges interfacing with previous andsubsequent purification steps, while eliminating need for anyintermediate step. It was determined that acetate buffer was the mostoptimal buffer to be used in this process.

DOE Method

A DoE (central composite) study was designed to characterize the loadratio, load conductivity, and load pH, while a second DoE (screening)was designed to characterize the process temperature, resin capacity,and process flow rate. Both DoE studies were designed using DesignExpert (version 8.0.6). Load ratio, load conductivity, and load pH werechosen as variables in the first DoE because earlier experiments showedthat they had an impact on product yield and product attributes. Processtemperature, resin capacity, and process flow rate were assessed in aseparate DoE because earlier studies showed that these parameters hadminimal effect on product yield and product attributes.

To perform regression analyses on the data, the results were enteredinto the Design Expert and analyzed first for normal distribution andoutliers via Box Cox and Externally Studentized Residuals, respectively.If the data was not normally distributed, transformations were conducted(e.g., log 10) based on the software's recommendations. Any outliersthat significantly weakened the models were omitted from the analysis.Afterwards, insignificant parameters (p-value >0.05) were removed usingthe backwards or manual elimination function to simplify the model. In afew cases, insignificant parameters were retained in the model tostrengthen the R2 and lack of fit. All models were built on theassumption that only main effects and 2-level interactions would besignificant and not be confounded.

Analytical Methods

The analytical methods utilized for all of the experiments aresummarized in Table 2. The starting material is sufficiently pure thatmeasurement of the product yield using OD280 method is accurate for theSE Hicap column.

TABLE 2 Summary of Analytical Methods Method Validation/ QualificationReport Analytical Method Measurement Unit Number(s) OD₂₈₀ Totalabsorbance in each fraction Non-dimensional Not Applicable and totalA₂₈₀ in the eluate Pressure Column inlet pressure Psig AKTA programUV₂₈₀ Chromatograph n/a Not Applicable HPLC-SEC Non-reversible solubleaggregate % TD-TDMP-9032 HPLC-HIC Peak 1, Peak 2, and Peak 3 %TD-TDMP-242 LC-90/GXII (Non- Purity and Highest Single Impurity %TD-TDMP-285 Reduced) LC-90/GXII Purity and LMW % TD-TDMP-286 (Reduced)HPLC-IEX Total Sialic Acid (TSA) Mole per mole TD-TDMP-370 iCIEF ChargedHeterogeneity % TD-TDMP-284 ECL Total Host Cell Protein Parts permillion TD-TDMP-252 (ppm) Q-PCR Total DNA Parts per billion TD-TDMP-321(ppb)

DOE 1 (Central Composite)

The DoE study characterizing load ratio, load pH, and load conductivitywas successfully completed without any deviations from the study design.A typical SE Hicap chromatogram under control conditions shows how theUV deflects with a pre-peak during the load, decreases in UV, and thenincreases in UV before reaching a plateau (FIG. 1). It was found thatweak protein-binding can lead to insufficient TSA enrichment (data notshown). It was found that strong protein-binding can lead to productloss (data not shown).

Low load pH and low load conductivity decreased product yield, enrichedTSA content, increased Peak 1 impurity, decreased aggregate level, andincreased monomer content (Table 3, see also Table 4). Load pH and loadconductivity have an interaction in that a low load pH can offset theeffects of high load conductivity on these outputs and vice versa. Asload conductivity or load pH increased, Peak 2 level decreased by theamount that was increased in Peak 3 level (Table 3, see also Table 4).This showed that varying load conductivity and load pH can change therelative distribution of Peaks 1, 2, and 3.

TABLE 3 DOE 1 (Central Composite) Data #1 Load Ratio Load Azso HMWReduced (OD/mL Conductivity Load Yield Aggregate Monomer Peak 1 Peak2Peak3 NANA NGNA TSA Purity Cycle resin) (mS/cm) pH (%) (%) (%) (%) (%)(%) (mol/mol) (mol/mol) (mol/mol) (%) 1 40.9 9.0 5.8 66.9 3.4 96.7 4.960.0 34.3 15.5 0.0 15.6 95.1 2 54.6 9.0 5.8 71.1 4.0 96.0 4.7 60.1 34.215.4 0.1 15.5 94.9 3 27.1 9.0 5.8 58.1 2.8 97.2 4.5 63.2 31.4 15.8 0.115.9 94.3 4 49.0 11.0 6.3 87.9 8.7 91.3 4.0 53.5 41.5 14.5 0.1 14.6 95.25 32.7 11.0 6.3 83.9 8.4 91.6 4.5 54.1 40.4 14.7 0.1 14.8 95.3 6 49.011.0 5.3 65.0 2.4 97.6 4.8 63.8 30.1 15.9 0.1 16.0 94.2 7 32.7 11.0 5.358.5 1.8 98.2 4.6 64.5 30.3 15.9 0.1 16.0 94.0 8 40.9 9.0 5.8 63.3 3.696.4 4.5 60.9 33.5 15.3 0.1 15.4 94.8 9 40.9 9.0 5.8 62.9 3.4 96.6 4.561.7 33.0 16.0 0.1 16.1 94.9 10 40.9 12.4 5.8 78.1 7.7 92.3 4.1 55.739.3 14.5 0.1 14.6 95.5 11 40.9 5.6 5.8 45.1 2.1 97.9 5.4 66.1 27.8 16.70.1 16.8 94.4 12 32.7 7.0 6.3 63.5 5.8 94.2 5.0 59.7 33.9 16.1 0.1 16.394.9 13 49.0 7.0 6.3 67.0 4.4 95.6 4.3 58.3 36.4 15.5 0.1 15.6 95.2 1449.0 7.0 5.3 28.5 1.8 98.2 7.4 70.5 20.5 17.7 0.1 17.8 92.3 15 32.7 7.05.3 13.7 0.0 100.0 12.9 67.3 16.5 18.5 0.1 18.6 86.3 16 40.9 9.0 5.021.5 0.0 100.0 9.9 70.8 17.0 18.2 0.1 18.3 89.7 17 40.9 9.0 6.6 82.9 8.291.8 4.5 54.5 39.9 14.8 0.1 14.9 95.2 18 40.9 9.0 5.8 67.6 3.6 96.4 4.062.9 32.5 15.9 0.1 16.1 95.1

TABLE 4 Effect of Load Ratio, Load pH and Load Conductivity on Yield,TSA, Aggregation, and Peak Size. Load Load Feed Ratio Load ConductivityA280 TSA Aggregate Peak Peak Peak Source (mg/mL) pH (mS/cm) Yield (%)(mole/mole) (%) 1 (%) 2 (%) 3 (%) C12-01 53 5.6 10 73.5 15.7 5.1 6.9 6132.1 C12-01 28 5.6 10 64.9 15.4 3.7 6.1 63.7 30.1 C12-01 56 5.6 10 74.615.7 5.1 6.2 60.6 33.2 ILS-003 53 5.8 11 78.5 15.4 8.1 5.8 63.1 31ILS-003 56 5.8 11 78.3 15.6 8.1 6 62.9 31.2 ILS-003 53 5.6 10 70.2 164.5 6.5 66.6 27 ILS-003 56 5.6 10 70.4 16.5 4.7 6.4 66.3 27.3

TABLE 5 DOE 1 (Central Composite) Data #2 Load Non- Ratio Load Non-Reduced HCP HCP DNA DNA Acidic Basic Main (OD/mL Conductivity LoadReduced Reduced Impurity Content Clearance Content Clearance IsoformIsoform Isoform Cycle resin) (mS/cm) pH LMW (%) Purity (%) (%) (ppm)(LRV) (ppb) (LRV) (%) (%) (%) 1 40.9 9.0 5.8 2.6 96.2 3.8 1581 0.3 58.41.5 57.7 5.3 37.1 2 54.6 9.0 5.8 2.6 96.5 3.5 1590 0.3 39.6 1.7 58.7 5.436.0 3 27.1 9.0 5.8 3.2 96.7 3.3 1721 0.3 44.4 1.7 60.4 4.7 34.8 4 49.011.0 6.3 2.4 96.7 3.3 1416 0.2 190.5 0.9 60.4 5.1 34.5 5 32.7 11.0 6.32.3 96.4 3.6 1327 0.3 175.2 0.9 59.9 4.7 35.4 6 49.0 11.0 5.3 3.1 96.43.6 1446 0.3 40.0 1.7 56.6 6.9 36.5 7 32.7 11.0 5.3 3.4 96.5 3.5 15160.4 24.4 2.0 56.1 6.2 37.7 8 40.9 9.0 5.8 2.8 96.6 3.4 1719 0.3 42.7 1.758.7 6.0 35.4 9 40.9 9.0 5.8 2.8 96.6 3.4 1698 0.3 54.2 1.6 58.6 5.036.4 10 40.9 12.4 5.8 2.2 97.1 2.9 1561 0.2 64.6 1.4 57.2 6.2 36.6 1140.9 5.6 5.8 3.7 96.7 3.3 2448 0.3 35.6 1.9 61.4 4.2 34.4 12 32.7 7.06.3 2.9 96.6 3.4 1834 0.3 86.0 1.4 59.2 5.2 35.7 13 49.0 7.0 6.3 2.791.5 8.5 1564 0.3 98.1 1.3 58.0 4.5 37.5 14 49.0 7.0 5.3 5.5 81.8 18.23024 0.4 11.7 2.6 63.4 4.4 32.2 15 32.7 7.0 5.3 11.0 88.4 11.6 6435 0.442.4 2.4 75.0 3.8 21.2 16 40.9 9.0 5.0 6.8 84.6 15.4 3644 0.4 21.3 2.566.5 5.0 28.5 17 40.9 9.0 6.6 2.2 96.8 3.2 1149 0.3 204.8 0.9 60.1 4.235.7 18 40.9 9.0 5.8 2.6 97.0 3.0 1387 0.3 39.1 1.7 57.0 5.2 37.8

A high load conductivity or high load pH decreased DNA clearance (Table5). However, load pH decreased DNA clearance to a greater extent thanconductivity. Also, a high load pH also decreased HCP clearance (Table5).

Low load pH and low load conductivity decreased purity level (relativeto all impurities) (Table 5). Load pH and load conductivity have aninteraction in that a low load pH can offset the effects of high loadconductivity on the purity level. As load conductivity or load pHincreased, the highest single impurity level and LMW decreased (Table5).

The negatively-charged acidic isoform was enriched at low conductivityand low pH, but reduced in concentration when the basic and mainisoforms desorbed from the column at high conductivity or high pH (Table5). Therefore, load pH and load conductivity have an interaction in thatload pH can offset the effects of load conductivity on the isoforms.

These experiments indicate that the operating space for SE Hicap shouldhave a load pH target of 5.6 with a range of 5.5-5.8, a loadconductivity target of 10.0 mS/cm with a range of 9.5-11 mS/cm, and aload ratio target of 41 mg/mL resin with a range of 33-54 mg/mL resin.These load conditions would ensure that the flow-through/wash pool haveTSA levels of 15-16.5 mole/mole, aggregate levels of <6%, Peak 1 levelsof <6%, DNA clearance of ˜1.5 LRV, and a sufficient product yield of55-80%.

DoE 2 (Screening)

The DoE study characterizing process temperature, resin capacity, andprocess flow rate was successfully completed without any deviations fromthe protocol. The data from this DoE study are listed in Table 6.Although the most significant variables were resin capacity and processtemperature, their impact on the product yield and product attributeswere negligible. Thus, the process is robust and can tolerate a wideoperating range for temperature, resin capacity, and flow rate.

TABLE 6 DOE 2 (Screening) Data. Process Resin Capacity Process HMWTemperature (mg Lysozyme/ Flow Rate A₂₈₀ Yield TSA Aggregate MonomerPeak 1 Peak 2 Peak 3 Cycle (° C.) mL resin) (cm/hr) (%) (mol/mol) (%)(%) (%) (%) (%) 1 26.0 125 75 76.8 14.6 4.6 95.4 4.2 63.8 32.0 2 26.0158 150 74.4 14.5 4.9 95.1 4.5 62.4 33.1 3 26.0 125 150 73.6 14.0 4.695.4 4.6 63.1 32.3 4 26.0 158 75 72.9 14.5 4.7 95.3 4.5 62.6 32.9 5 16.0125 150 69.1 14.4 4.1 95.9 4.2 64.7 31.1 6 16.0 158 75 71.0 14.8 4.295.8 4.3 64.3 31.4 7 16.0 158 150 70.0 14.7 4.3 95.7 4.3 64.4 31.3 816.0 125 75 68.7 14.4 4.1 95.9 4.5 65.0 30.5 9 21.0 142 113 72.4 14.74.3 95.7 4.9 64.2 30.9 10 21.0 142 113 71.5 14.7 4.3 95.7 4.9 64.2 30.911 21.0 142 113 72.4 13.6 4.4 95.6 4.9 64.6 30.5 12 21.0 142 113 71.514.5 4.3 95.7 5.1 64.1 30.8

Conclusions

The SE Hicap flow-through chromatography was successfully developed asthe second purification step to enrich TSA levels to 15-16.5 mole/mole,decrease aggregates by ˜12%, and reduce DNA by ˜1.5 LRV, whilemaintaining high product recoveries of 55-75% (A280) under targetoperating conditions. To meet these key and critical product attributes,the recommended operating space for SE Hicap would have a load pH targetof 5.6±0.2, a load conductivity target of 10.0 mS/cm with a range of9.5-11 mS/cm, and a load ratio target of 41 mg/mL resin±8 mg/mL resinbased on DoE results. Other process parameters such as processtemperature, process flow rate, and resin capacity had negligible impacton product yield and product attributes.

Example 2 Enrichment of TSA Using CEX Resin

Having determined the enrichment parameters for the SE Hicap CEX resin,the operating conditions necessary to enrich TSA while removingaggregates and/or misfolds was determined for other CEX resins. Theresins tested were: GE SPXL, GE SP Sepharose, Millipore Eshmuno S, TosohGigacap CM, Tosoh Gigacap S-650, and BioRad Nuvia S. The final operatingconditions for each column can be seen below in Table 7:

TABLE 7 Operating Conditions for CEX Columns. TSA Misfolds ConductivityEnriched Aggregate Removed Columns pH (mS/cm) Yield (%) (mole/mole)Removed (%) (%) SE Hicap 5.6 10 47.9 3.1 21.2 15.8 Control SP Sepharose5 9 44.0 2.3 9.5 26.8 SP Sepharose 5 6 53.3 2.2 7.3 16.0 XL Nuvia S 5 947.6 3.8 17.6 12.7 Eshmuno S 5 9 55.0 2.5 16.7 13.7 GigaCap S 5 8 46.93.9 20.3 13.7 GigaCap CM 5 6 40.1 4.0 22.5 20.7

A comparison of the enrichment of TSA by each column, with the resultsnormalized to 50% of product yield, can be seen in FIG. 2. A comparisonof the aggregates and misfolds removed by each column, with the resultsnormalized to 50% of product yield, can be seen in FIG. 3.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method for enriching the level ofpost-translational modification of recombinant polypeptides, comprising:contacting a composition which comprises an initial population ofrecombinant polypeptides having different levels of post-translationalmodification with a cation exchange chromatography (CEX) medium operatedin a flow-through mode; wherein recombinant polypeptides that do notbind the CEX medium are separated from recombinant polypeptides thatbind the CEX medium, and wherein the recombinant polypeptides that donot bind the CEX medium comprise a higher level of post-translationalmodification compared to the bound recombinant polypeptides.
 2. A methodfor enriching the level of post-translational modification ofrecombinant polypeptides, comprising: a) contacting a composition whichcomprises an initial population of recombinant polypeptides havingdifferent levels of post-translational modification with a cationexchange chromatography (CEX) medium operated in a flow-through mode;and b) separating recombinant polypeptides that do not bind the CEXmedium from recombinant polypeptides that bind the CEX medium; whereinthe recombinant polypeptides that do not bind the CEX medium that arerecovered comprise a higher level of post-translational modificationcompared to the bound recombinant polypeptides.
 3. A method forenriching the level of post-translational modification of recombinantpolypeptides, comprising: a) contacting a composition which comprises aninitial population of recombinant polypeptides having different levelsof post-translational modification with a cation exchange chromatography(CEX) medium operated in a flow-through mode; b) separating recombinantpolypeptides that do not bind the CEX medium from recombinantpolypeptides that bind the CEX medium; and c) recovering the recombinantpolypeptides that do not bind the CEX medium; wherein the recombinantpolypeptides that do not bind the CEX medium comprise a higher level ofpost-translational modification compared to the bound recombinantpolypeptides.
 4. A method for enriching the level of post-translationalmodification of recombinant polypeptides, comprising: a) providing acomposition which comprises an initial population of recombinantpolypeptides having different levels of post-translational modification;b) contacting the composition with a cation exchange chromatography(CEX) medium operated in a flow-through mode; c) separating recombinantpolypeptides that do not bind the CEX medium from recombinantpolypeptides that bind the CEX medium; and d) recovering the recombinantpolypeptides that do not bind the CEX medium; wherein the recombinantpolypeptides that do not bind the CEX medium comprise a higher level ofpost-translational modification compared to the bound recombinantpolypeptides.
 5. The method of claim 1 or 2, further comprisingrecovering the recombinant polypeptides that do not bind the CEX medium.6. The method of any one of claims 1-5, wherein the post-translationalmodification is sialylation or gamma-carboxyglutamate (Gla) formation.7. The method of claim 6, wherein the post-translational modification issialylation.
 8. The method of claim 7, wherein the total sialic acidcontent of the recombinant polypeptides that do not bind the CEX mediumis between about 0.5 and about 6 moles of sialic acid per mole ofprotein higher than that of the initial population of recombinantpolypeptides.
 9. The method of claim 8, wherein the total sialic acidcontent of the recombinant polypeptides that do not bind the CEX mediumis between about 0.5 and about 4 moles of sialic acid per mole ofprotein higher than that of the initial population of recombinantpolypeptides.
 10. The method of claim 7, wherein the total sialic acidcontent of the recombinant polypeptides that do not bind the CEX mediumis between about 5% and about 100% higher than that of the initialpopulation of recombinant polypeptides.
 11. The method of claim 10,wherein the total sialic acid content of the recombinant polypeptidesthat do not bind the CEX medium is between about 5% and about 40% higherthan that of the initial population of recombinant polypeptides.
 12. Themethod of any one of claims 7-11, wherein the total sialic acid contentof the recombinant polypeptides that do not bind the CEX medium isbetween about 12 and about 20 moles of sialic acid per mole of protein.13. The method of claim 12, wherein the total sialic acid content of therecombinant polypeptides that do not bind the CEX medium is betweenabout 14 and about 17 moles of sialic acid per mole of protein.
 14. Themethod of any one of claims 7-10, wherein the total sialic acid contentof the initial population of recombinant polypeptides is about 10 toabout 14 moles of sialic acid per mole of protein.
 15. The method ofclaim 14, wherein the total sialic acid content of the initialpopulation of recombinant polypeptides is about 13-14 moles of sialicacid per mole of protein.
 16. The method of any one of claims 1-15,wherein the recombinant polypeptide comprises an Fc domain.
 17. Themethod of claim 16, wherein the recombinant polypeptide comprises anantibody.
 18. The method of claim 16, wherein the recombinantpolypeptide comprises an Fc fusion polypeptide comprising a ligandbinding domain of a receptor.
 19. The method of claim 18, wherein thereceptor is a TNF receptor.
 20. The method of claim 19, wherein therecombinant polypeptide is etanercept.
 21. The method of any one ofclaims 1-18, wherein the recombinant polypeptide comprises a clottingfactor.
 22. The method of claim 21, wherein the recombinant polypeptideis a monomer-dimer hybrid.
 23. The method of claim 21 or 22, wherein theclotting factor is selected from Factor VII (FVII), FVIIa, Factor VIII(FVIII), Factor IX (FIX), or FIXa (FIX).
 24. The method of claim 23,wherein the FVIII is full-length FVIII or B-domain deleted FVIII. 25.The method of claim 23 or 24, wherein the FVIII is single chain FVIII ordual chain FVIII.
 26. The method of any one of claims 1-25, wherein thecontacting occurs at a load ratio between about 30 and about 100 mgtotal protein/ml CEX medium.
 27. The method of claim 26, wherein thecontacting occurs at a load ratio between about 33 and about 54 mg totalprotein/ml CEX medium.
 28. The method of claim 27, wherein thecontacting occurs at a load ratio of at least about 41 mg totalprotein/ml CEX medium.
 29. The method of any one of claims 1-28, whereinthe contacting occurs at a pH between about 4 and about
 7. 30. Themethod of claim 29, wherein the contacting occurs at a pH between about5 and about
 6. 31. The method of claim 30, wherein the contacting occursat a pH between about 5.5 and about 5.8
 32. The method of claim 30,wherein contacting occurs at a pH of at least about 5.6.
 33. The methodof any one of claims 1-32, wherein the contacting occurs at aconductivity between about 8 and about 12 mS/cm.
 34. The method of claim33, wherein the contacting occurs at a conductivity of between about 9.5and about 11 mS/cm.
 35. The method of claim 34, wherein the contactingoccurs at a conductivity of at least about 10 mS/cm.
 36. The method ofany one of claims 1-35, wherein the recombinant polypeptides that do notbind the CEX medium comprise about 25% to about 80% of the initialpopulation of recombinant polypeptides.
 37. The method of claim 36,wherein the recombinant polypeptides that do not bind the CEX mediumcomprise about 55% to about 80% of the initial population of recombinantpolypeptides.
 38. The method of any one of claims 1-37, wherein the CEXmedium comprises a ligand selected from the group consisting ofsulfoethyl; sulphopropyl; sulfopropyl; CH₂—SO₃ ⁻; CH₂CH₂CH₂SO₃ ⁻; SO₃ ⁻;and CH₂—COO⁻¹.
 39. The method of claim 38, wherein the CEX mediumcomprises a sulfoethyl ligand.
 40. The method of claim 39, wherein theCEX medium comprises a binding capacity of between about 120 and about160 mg lysozyme/ml resin.
 41. The method of any one of claims 1-40,wherein the recombinant polypeptide is produced by a eukaryotic hostcell.
 42. The method of claim 41, wherein the eukaryotic host cell is amammalian host cell.
 43. The method of any one of claims 1-42, whereinthe contacting is performed at a manufacturing scale.
 44. The method ofany one of claims 1-43, wherein the composition further comprises atleast one impurity.
 45. The method of claim 44, wherein the impuritycomprises a DNA, RNA, lipid or protein.
 46. The method of claim 45,wherein the impurity comprises a protein.
 47. The method of claim 46,wherein the protein impurity comprises a truncated form of therecombinant polypeptide, an aggregated form of the recombinantpolypeptide, or a misfolded form of the recombinant polypeptide.
 48. Themethod of any one of claims 44-47, providing a final compositioncomprising the recombinant polypeptides that do not bind the CEX medium,wherein the final composition comprises less impurities than thecomposition that comprised the initial population of polypeptides.